Papillomavirus infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes and cows. Papillomaviruses infect epithelial cells, generally inducing benign epithelial or fibroepithelial tumors at the site of infection. Papillomaviruses are species specific infective agents; a human papillomavirus cannot infect a nonhuman animal.
Papiromaviruses may be classified into distinct groups based on the host that they infect. Human papillomaviruses (HPV) are further classified into more than 60 types based on DNA sequence homology (for a review, see Papillomaviruses and Human Cancer, H. Pfister (ed.), CRC Press, Inc., 1990). Papillomavirus types appear to be type-specific immunogens in that a neutralizing immunity to infection to one type of papillomavirus does not confer immunity against another type of papillomavirus.
In humans, different HPV types cause distinct diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts in both normal and immunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25, 36 and 46-50 cause flat lesions in immunocompromised individuals. HPV types 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata of the genital or respiratory mucosa. HPV types 16 and 18 cause epithelial dysplasia of the genital mucosa and are associated with the majority of in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal. HPV6 and HPV11 are the causative agents for more than 90% of all condyloma (genital warts) and laryngeal papillomas.
Immunological studies in animals have shown that the production of neutralizing antibodies to papillomavirus antigens prevents infection with the homologous virus. The development of effective papillomavirus vaccines has been slowed by difficulties associated with the cultivation of papillomaviruses in vitro. The development of an effective HPV vaccine has been particularly slowed by the absence of a suitable animal model. Neutralization of papillomavirus by antibodies appears to be type-specific and dependent upon conformational epitopes on the surface of the virus.
Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode for up to eight early and two late genes. The open reading frames (ORFs) of the virus genomes are designated E1 to E7 and L1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 code for virus capsid proteins. The early (E) genes are associated with functions such as viral replication and cellular transformation.
The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. L2 protein is a minor capsid protein which has a predicted molecular weight of 55-60 kDa and an apparent molecular weight of 75-100 kDa as determined by polyacrylamide gel electrophoresis. Immunologic data suggest that most of the L2 protein is internal to the L1 protein. The L2 proteins are highly conserved among different papillomaviruses, especially the 10 basic amino acids at the C-terminus. The L1 ORF is highly conserved among different papillomaviruses.
The L1 and L2 genes have been used to generate vaccines for the prevention and treatment of papiromaviruses infections in animals. Zhou et al., (1991; 1992) cloned HPV type 16 L1 and L2 genes into a vaccinia virus vector and infected CV-1 mammalian cells with the recombinant vector to produce virus-like particles (VLP).
Recombinant baculoviruses expressing HPV6 L1, HPV11 L1, HPV16 L1, HPV18 L1, HPV31 L1 or HPV16 L2 ORFs have been used to infect insect Sf9 cells and produce L1 and L2 proteins. Western blot analyses showed that the baculovirus-derived L1 and L2 proteins reacted with antibody to HPV16. The baculovirus derived L1 forms VLPs.
Carter et al., (1991) demonstrated the production of HPV 16 L1 and HPV16 L2 proteins by recombinant strains of Saccharomvces cerevisiae. Carter et al. also demonstrated the production of HPV6b L1 and L2 proteins. The HPV6b L1 protein was not full-length L1 protein. The recombinant proteins were produced as intracellular as well as secreted products. The recombinant L1 and L2 proteins were of molecular weights similar to the native proteins. When the proteins were expressed intracellularly, the majority of the protein was found to be insoluble when the cells were lysed in the absence of denaturing reagents. Although this insolubility may facilitate purification of the protein, it may hamper analysis of the native epitopes of the protein.
Recombinant proteins secreted from yeast were shown to contain yeast-derived carbohydrates. The presence of these N-linked oligosaccharides may mask native epitopes. In addition, the secreted recombinant proteins may contain other modifications, such as retention of the secretory leader sequence.
The present invention is directed to the production of recombinant papillomavirus proteins having the immunity-conferring properties of the native papillomavirus proteins as well as methods for their production and use. The present invention is a series of synthetic virus-like particles useful in the characterization of human papillomavirus infection and assays employing the synthetic virus-like particles.
The invention involves the delineation of residues specific to HPV11 L1 which are required for binding neutralizing antibodies, and a modified HPV16 L1 gene with HPV11-like substitutions such that VLPs produced from the modified HPV16 L1 gene also bind HPV11 neutralizing monoclonal antibodies.
We previously demonstrated that HPV11 L1 residues Gly.sup.131 -Tyr.sup.132 were responsible for the HPV11 specificity of binding of several HPV11 neutralizing monoclonal antibodies. Because the binding of these antibodies is conformationally dependent, it remained unanswered as to whether the epitope is continuous and comprised of residues located next to each other with conformation requiring VLP assembly, or discontinuous and comprised of residues well separated on the L1 linear sequence but which come into close proximity upon proper folding and assembly of particles. We scanned residues over a 20 residue stretch centered at Gly.sup.131 -Tyr.sup.132, and identified five residues where substitution resulted in significant loss of binding of the neutralizing monoclonal antibodies, without affect on other HPV11 specific, VLP-dependent antibodies. This demonstrates that the epitope is continuous. This was confirmed by demonstrating that HPV11 substitutions at these positions into the HPV16 L1 sequence forms the basis of transfer of binding of these monoclonal antibodies to modified HPV16 VLPs.
The panel of neutralizing monoclonal antibodies for HPV11 was obtained from Neil Christensen (Pennsylvania State University, Hershey, Pa). The monoclonal antibodies in the panel are HPV11 specific and VLP-dependent. The antibodies may be distinguished from each other in terms of which amino acid residues affect binding of the individual antibodies, although there are overlapping positions for all the monoclonal antibodies. Additional antibodies used in these studies were also obtained from Dr. Neil Christensen.
These residues collectively define the epitope for antibodies known to neutralize HPV11 . We also demonstrate that substitution of these residues into equivalent positions of the HPV16 L1 sequence form the basis of transferring binding of these antibodies to modified HPV16 VLPs. The modified HPV16 VLPs may be used to develop HPV11 specific serological assays. Because of the high identity between HPV6 and HPV11 L1 sequences, present serological assays cannot distinguish responses between these two types very well. Modified HPV16 VLPs with a single HPV11 specific epitope and no cross-reactivity to HPV6 VLPs should be able to identify HPV11 immune responses upon infectivity or immunization.
This problem has not been solved in the past and, to our knowledge, is the first demonstration that a conformationlly dependent epitope is continuous.
There were two difficulties to overcome. First, the epitope is conformational, and conventional means of epitope mapping, binding to peptide fragments, could not be utilized. It was necessary to express any test L1 protein in a way that facilitated formation of virus-like particles which minic the virus structure. Second, the large number of L1 clones required for the mapping necessitated the generation of a facile means to express the test viral coat proteins.
Without isolation of a type-specific epitope, it would be difficult to distinguish HPV6 and HPV11 immune responses.
One use of the derivatized HPV16 VLP is as a reagent in a serological assay. Because most epitopes are shared between HPV6 and HPV11 VLPs, polyclonal sera to one competes with the binding of a type-specific monoclonal antibody to the other due to steric hindrance from the binding of antibodies to neighboring sites. There are very few cross-reactive epitopes between HPV16 and either HPV11 or HPV6. Therefore, presentation of an HPV11 specific epitope on an HPV16 VLP should eliminate the problem of steric competition from neighboring epitopes. Only the presence of antibody in a polyclonal response to the specifically transferred epitope should compete with monoclonal antibody binding.